Significant progress has been made in the development of a live attenuated subgroup A RSV vaccine as well as in the understanding of the genetic basis of attenuation of the biologically-derived, live-attenuated virus vaccine candidates. Toward this goal, a set of five missense mutations previously identified by nucleotide sequence analysis of subgroup A cold- passaged (cp) respiratory syncytial virus (RSV) have been introduced into a recombinant wildtype strain of RSV. This recombinant virus, designated rA2cp, caused significantly less rhinorrhea and cough and replicated less efficiently in the upper and lower respiratory tracts of seronegative chimpanzees than wildtype RSV. These findings confirm the role of the cp mutations in attenuation of RSV and identify their usefulness for inclusion in future live attenuated recombinant RSV vaccine candidates. Respiratory syncytial virus (RSV) cpts248/404 is a live- attenuated, temperature-sensitive (ts) vaccine candidate derived from cold-passaged cpRSV by two rounds of chemical mutagenesis and biological selection. In addition to the cp mutations, the cpts248/404 candidate vaccine possessed the following mutations in L: Gln-831-Leu (248 mutation) and Asp-1183-Glu (404-L mutation). A third mutation resulted in a nucleotide substitution at position 9 of the cis-acting gene start signal of the M2 gene (404-M2 mutation). In the present study, the genetic basis of attenuation of cpts248/404 was defined by the introduction of each of these mutations (singly or in combination) into a full-length cDNA clone of cpRSV. The 248 and 404-M2 mutations each specified the ts and attenuation (att) phenotypes. Thus, the cpts248/404 virus contains a set of ts and non-ts (i.e., the cp mutations) attenuating mutations, which likely accounts for its high level of genetic stability. The recombinant version of this virus, rA2cp248/404, was phenotypically indistinguishable from cpts248/404 and represents a background into which additional mutations can be introduced as needed to obtain the desired level of attenuation for successful immunization of the very young human infant. cpts530/1009 is a live-attenuated, temperature-sensitive (ts) RSV vaccine candidate that was shown previously to be attenuated for seronegative humans. The 530 and1009 mutations each contributed to the ts and att phenotypes and their effects were additive. These data show that the genetic basis of the attenuation and temperature sensitivity of the cpts530/1009 candidate vaccine virus is the sum of the contributions of seven identified amino acid substitutions, i.e., the 5 cpRSV mutations, the 530 mutation, and the 1009 mutation.